LSC - 2024 - Multiplex protein quantification per cell type in bronchoalveolar lavage cytospin cells of patients with asthma and healthy individuals

Bronchoalveolar lavage (BAL) cells provide some of the most valuable and targetable immune profile information for patients with asthma. Therefore, we developed a method to quantify a multiplex of proteins that also considers the four primary cell types of BAL using NanoStrings GeoMx. Overall, 21 matched BAL cytospins from the BREATHE cohort (Healthy: n=8, Mild-Asthma: n=7, Severe-Asthma: n=6) underwent morphology staining to identify the macrophages, neutrophils and eosinophils. Lymphocytes were identified as ‘the rest’ of the SYTO-13 stained nuclei. Three protein panels assessing the immune profile, immune cell types and cell death markers were combined to quantify 40 proteins per cell type per patient. The most significant initial analysis results (p<0.0001) suggests Healthy BAL macrophages have significantly higher levels of ribosomal kinase S6 (S6), programmed death ligand-1 (PD-L1) and the cytokeratin’s (PanCK) than Asthma (n=13). Healthy macrophages tended to have more S6, MHC class II receptor (HLA-DR) and CD11c than Mild-Asthma but not Severe-Asthma. All Severe-Asthma cell types had the highest levels of Ki-67. Additionally, neutrophils, eosinophils and lymphocytes from Severe-Asthma all tended to have higher levels of Beta-2-microglobulin and B-cell lymphoma 6 (BCL6) (and CD20 on neutrophils only) than Healthy or Mild-Asthma. To our knowledge, this is the first high-plex protein profiling of each BAL cell type comparing patients with asthma to healthy individuals. Results thus far highlight that, despite having the same cell types present in each BAL, their current and potential activities differ considerable between these three groups.